The use of antibiotics for the treatment of infectious diseases is compromised due to the increase in the number of antibiotic-resistant bacterial strains. Thus, multi-resistant bacteria are a major health problem and the need for new antibiotics is apparent. In this invention a novel combination of silver ions with selenazol and thiazolone drugs targeting the thioredoxin system has been discovered. The strong synergistic effects observed will allow a wide range of bacteria to be targets of this new drug.
Thioredoxin
The thioredoxin (Trx) system and the GSH-glutaredoxin (Grx) system are two major thiol dependent disulfide reductases in the cells, which transfer the electrons from NADPH to their substrates (1-3). The two thiol dependent electron transferring pathways play critical roles in defense against oxidative stress by reducing methionine sulfoxide reductases (MSR) to repair proteins or peroxiredoxins (Prx) to remove peroxides. They are also electron donors for ribonucleotide reductase (RNR), which is essential for the production of deoxyribonucleotides and DNA (FIG. 6).
The thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH are together called the thioredoxin system, which serves as a hydrogen donor for ribonucleotide reductase and has a general powerful disulfide reductase activity (4, 5, 11, 13). The thioredoxin system is present in cells and in all forms of life (4, 5, 11, 13). Thioredoxin reductase (TrxR) is a dimeric FAD containing enzyme that catalyzes the reduction of its main protein substrate oxidized thioredoxin, to reduced thioredoxin at the expense of NADPH. The enzyme mechanism involves the transfer of reducing equivalents of NADPH to a redox active site disulfide via an FAD domain. Thioredoxin reductase from Escherichia coli with subunits of 35 kDa has been extensively characterized (46). X-ray crystal structure reveals that the active site disulfide is located in a buried position in the NADPH domain (22) and suggests that it should undergo a large conformational change to create a binding site for Trx-S2 and reduction by a dithiol-disulfide exchange.
Trx system is composed with thioredoxin reductase (TrxR), Trx and NADPH. Trx is ubiquitous in all living organisms with its conserved CGPC active site and the Trx fold (1). In contrast, the TrxRs in mammalian cells and bacteria showed notable differences in structure and reaction mechanism (4-6). Bacteria have a smaller (70 kDa) sulfur-dependent enzyme whereas human and animal cells have a large (115 kDa) selenocysteine-containing enzyme (1-3,6). Moreover, many pathogenic bacteria contain distinct thiol-dependent redox systems (7). Particularly, some pathogenic bacteria lack glutathione (GSH) and glutaredoxin (Grx) and thus TrxR and Trx are essential for DNA synthesis and the Trx system should be a suitable target for development of antibacterial drugs (4, 8, 9).
Thioredoxin reductase is a ubiquitous enzyme present in all cells. However, the enzyme is often over-expressed in tumor cells compared to normal tissues, and tumor proliferation seems to be crucially dependent on an active thioredoxin system, making it a potential target for anticancer drugs (16). Over the last decade a number small organic and organometallic molecules that include platinum and gold containing complexes (47-50) naphthoquinone spiroketal based natural products (51-53), different naphthazarin derivatives (54), certain nitrosoureas (55-56) and general thiol (or selenol) alkylating agents such as 4-vinylpyridine, iodoacetamide, or iodoacetic acid (57) have been identified as inhibitors of Trx or TrxR or both. Engman et al. have reported the inhibition of mammalian thioredoxin reductase by diaryldichalcogenides (58) and organotellurium compounds (59-61). However, no inhibition has been presented for bacterial TrxR.
Thioredoxins together with glutaredoxins are the two dithiol hydrogen donors for the essential enzyme ribonucleotide reductase required for DNA synthesis (FIG. 6) (4, 5). As shown in FIG. 6 the two enzymes glutathione reductase (GR encoded by the gor gene) and thioredoxin reductase (TrxR encoded by the trxB gene) in E. coli are central in electron transport from NADPH (6). Thioredoxin reductase from human and animal cells is a large selenoenzyme and very different from the enzymes present in all prokaryotes (7, 8). In contrast to the mammalian enzymes the E. coli enzyme is highly specific and utilizes a different mechanism with an involvement of protein conformation change as mentioned above (9).
Thioredoxin reductase (TrxR), catalyzes the electron donation from NADPH via thioredoxin (Trx) to ribonucleotide reductase (RNR) and may be essential for DNA synthesis if no other system is present. Cytosolic Trx is a highly conserved 12 kDa protein whereas the cytosolic TrxRs from mammalian and bacterial, e.g. Escherichia coli, are very different in their structure and catalytic mechanisms, with mammalian TrxR being a large selenoenzyme.
Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one is an antioxidant and anti-inflammatory selenoorganic compound (1) used in clinical trials against e.g. stroke (2). It is thus known to be safely administered to humans. Ebselen and ebselen diselenide have been reported as substrates for mammalian thioredoxin reductase (3a) and its reaction mechanisms have been published (3b, 32). There are several reports of synthesis of substituted benzisoselenazol-3(2H)-ones. Some of these compounds were reported as inhibitors of viral cytopathogenicity and active immunostimulants inducing cytokines, such as interferons (IFNs), tumor necrosis factors (TNFs) and interleukin (IL-2) in human peripheral blood leukocytes (62-64). However, none of the reports indicates thioredoxin reductase activity.
It has been shown that ebselen, which has been known as a glutathione peroxidase (GSPx) mimic (1), is a substrate for human and mammalian thioredoxin reductase and a highly efficient oxidant of reduced thioredoxin (3a,3b). This strongly suggested that the thioredoxin system (NADPH, thioredoxin reductase and thioredoxin) is the primary target of ebselen, since a highly efficient reduction of hydroperoxides was given by ebselen in the presence of the thioredoxin system (3).
The cyclic-di-GMP (cdiGMP) signaling pathway regulates biofilm formation, motility, and pathogenesis. Pseudomonas aeruginosa is an important opportunistic pathogen that utilizes cdiGMP-regulated polysaccharides, including alginate and pellicle polysaccharide (PEL), to mediate virulence and antibiotic resistance. CdiGMP activates PEL and alginate biosynthesis by binding to specific receptors including PelD and Alg44. Ebselen was identified as an inhibitor of cdiGMP binding to receptors containing an RxxD domain including PelD and diguanylate cyclases (DGC). Ebselen reduces diguanylate cyclase activity by covalently modifying cysteine residues. Ebselen oxide, the selenone analogue of ebselen, also inhibits cdiGMP binding through the same covalent mechanism. Ebselen and ebselen oxide inhibit cdiGMP regulation of biofilm formation and flagella-mediated motility in P. aeruginosa through inhibition of diguanylate cyclases. Lieberman, O. J. et al. “High-Throughput Screening Using the Differential Radial Capillary Action of Ligand Assay Identifies Ebselen As an Inhibitor of Diguanylate Cyclases”, ACS Biology 2014, 9, 183-192.
Ebselen and Derivatives
It was previously discovered that that ebselen [2-phenyl-1,2 benzisoselenazol-3(2H)-one], (EbSe) which is a substrate of mammalian TrxR and an competitive reversible inhibitor of bacterial TrxR, displays selective antibacterial activity toward certain bacteria lacking glutathione (48). The pathogenic bacteria including Helicobacter pylori, Mycobacterium tuberculosis, and Staphyloccus aureus exhibit high sensitivity to ebselen (48).
The thioredoxin (Trx), thioredoxin reductase (TrxR), and NADPH are together called the thioredoxin system, which serves as a hydrogen donor for ribonucleotide reductase and has the most general powerful disulfide reductase activity. (4, 5, 11, 13) The thioredoxin system is present in cells and in all living systems. (4, 5, 11, 13) Thioredoxin reductase (TrxR) is a dimeric FAD containing enzyme that catalyzes the reduction of its main protein substrate oxidized thioredoxin, to reduced thioredoxin at the expense of NADPH. The enzyme mechanism involves the transfer of reducing equivalents of NADPH to a redox active site disulfide via FAD domain. Thioredoxin reductase from Escherichia coli with subunits of 35 kDa has been extensively characterized. (46) X-ray crystal structure reveals that the active site disulfide is located in a buried position in the NADPH domain, (22) and suggests that it should undergo a large conformational change to create a binding site for Trx-S2 and reduction by a dithiol-disulfide exchange.
Thioredoxin reductase is a ubiquitous enzyme present in all living cells. However, the enzyme is often over-expressed in tumor cells compared to normal tissues, and tumor proliferation seems to be crucially dependent on an active thioredoxin system, making it a potential target for anticancer drugs. (16) Over the last decade a number small organic and organometallic molecules that include platinum and gold containing complexes, (47, 48, 49, 50) naphthoquinone spiroketal based natural products, (51, 52, 53) different naphthazarin derivatives, (54) certain nitrosoureas, (55, 56) and general thiol (or selenol) alkylating agents such as 4-vinylpyridine, iodoacetamide, or iodoacetic acid (57) have been identified as inhibitors of Trx or TrxR or both. Engman et al. have reported the inhibition of thioredoxin reductase by diaryldichalcogenides (58) and organotellurium compounds. (60, 61)
Ebselen and ebselen diselenide have been reported as substrates for mammalian thioredoxin reductase and its reaction mechanism. (32, 77) Using glutathione as the reductant, the H2O2 reductase activity of ebselen was compared with that in the presence of the mammalian thioredoxin system. Formation of ebselen diselenide may serve as a dose-dependent storage form of ebselen, which can be relatively slowly activated to the catalytically active selenol by the mammalian thioredoxin system. The studies were extended to E. coli TrxR, and surprisingly, ebselen was found to inhibit E. coli TrxR. These findings lead to a search for the new organoselenium compounds containing the basic structure of ebselen, to study their reactivity with thioredoxin reductase.
There are several reports of synthesis of substituted benzisoselenazol-3(2H)-ones. Some of these compounds were reported as inhibitors of viral cytopathogenicity and active immunostimulants inducing cytokines, such as interferons (IFNs), tumor necrosis factors (TNFs) and interleukin (IL-2) in human peripheral blood leukocytes. (62, 63, 64) 2-(4-caroboxyphenyl)benzisoselenazol-3(2H)-one was found to be potent and selective inhibitor of endothelial nitric oxide synthase. (78). However, none of the reports indicates thioredoxin reductase activity.
Ebselen, a small isoselenazol drug well known for its antioxidant and anti-inflammatory properties, also has antibacterial properties. The mechanism has been unknown and there is a remarkable difference in sensitivity between Staphyloccus aureus being a 100-fold more sensitive than E. coli (10). The growth of methicillin resistant Staphylococcus aureus was shown to be inhibited by 0.20 μg per ml of ebselen, whereas strains of Enterobacteriaceae like E. coli NHHJ were much more resistant requiring up to 50 μg per ml. The MIC for 90% of S. aureus strains was 1.56 μg per ml and the drug was bacteriocidal (10).
Control of bacterial infection using chemotherapeutic principles and antibiotics are based on inhibition of cell wall synthesis, protein synthesis and other metabolic pathways. The presently used drugs have limitations and resistant bacterial infections is an increasing problem as evident by development of vancomycin and methicillin resistant bacteria. Since genetic material in the form of DNA is common to all microorganisms, inhibition of DNA synthesis is an attractive principle. In addition, drugs interrupting the defense of bacteria against oxidative stress should be a useful principle for developing new antibacterial agents.
The thioredoxin system, including thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, is the most powerful protein disulfide reductase in cells (4, 5, 11-13). Together with the glutaredoxin system, including glutaredoxin (Grx), glutathione (GSH), glutathione reductase (GR) and NADPH, thioredoxins are important hydrogen donors of ribonucleotide reductase for DNA synthesis and play key roles in cell redox regulation and growth control (4-6, 12, 14).
Thioredoxin reductase is one of those few examples of enzymes where the same reaction is catalyzed by more than one structure and mechanism (9, 15). Extensive studies on the features and redox properties of TrxR from various organisms resulted in the classification of two TrxRs, one from higher eukaryotes with high molecular weight and structurally resembles the other oxidoreductases; the other from prokaryotes, fungi, and plants with low molecular weight and distinct in structures and catalytic mechanism. Thus the striking difference between the enzymes would make them ultimate targets for novel antibiotic drug designs (16) although this has not yet been reported.
The TrxR from mammalian is a large selenoprotein with homodimer of 55 kD per subunits and a structure closely related to glutathione reductase but with an elongation containing a catalytically active selenol-thiol/selenosulfide in the conserved C-terminal sequence Gly-Cys(496)-Sec(497)-Gly, and thus a wide substrate specificity (7, 8, 15, 17-19). The bacterial counterpart of TrxR is however a non-selenolprotein with homodimer of 35 kD per subunits (9, 20, 21). Each E. coli TrxR monomer consists of an NADPH-binding domain and an FAD binding domain connected by a double-stranded ß-sheet. The active site Cys (135)-Ala-Thr-Cys(138) is located in the NADPH domain. A well-recognized characteristic of the E. coli enzyme is its large conformational change during catalysis. In its 3-D structure, the flow of electrons from NADPH to the active-site disulfide via the flavin can only be possible if the NADPH domain graphically rotating over 67° relative to the FAD domain, allowing an efficient hydride transfer from NADPH to FAD (the nicotinamide ring and the isoalloxazine would be in close contact) and simultaneously exposing the redox-active disulphide to the surface of the protein, accessible for the substrate (22, 23). Mammalian TrxRs are large dimeric selenoproteins (Mr 114.000), with structures closely related to glutathione reductase, but with a C-terminal 16 amino acid elongation containing a unique catalytically active conserved sequence Gly-Cys-Sec-Gly. Mammalian thioredoxin reductases have a remarkably wide substrate specificity. E. coli TrxR is smaller (Mr 70.000/dimer), with the active-site Cys-Ala-Thr-Cys disulfide loop located in the NADPH domain. During catalysis, a large conformational change is required, i.e., from FO (flavin oxidation by disulphide) to FR (flavin reduction by NADPH) form as discussed above.
Ribonucleotide reductase is a universal enzyme, which for aerobic organisms supply all four deoxyribonucleotides required for DNA synthesis de novo, for either replication or repair (FIG. 6). Electrons for the reduction ultimately are from NADPH via either thioredoxin or glutaredoxin. These two small protein thiol electron donors are reduced by separate pathways. Thioredoxin is reduced by thioredoxin reductase, and glutaredoxin by the tripeptide glutathione (GSH), which is present in high millimolar concentrations in most cells. Oxidized glutathione (GSSG) is reduced by glutathione reductase.
Whereas, there are general overall similarities between thioredoxin, glutaredoxin and ribonucleotide reductase in bacteria and human and other mammalian cells, there are fundamental differences between thioredoxin reductase enzymes. Thus, the enzyme is by convergent evolution either low molecular weight specific enzymes like that in E. coli or other bacteria or a high molecular weight selenocysteine-containing enzyme with broad specificity like the three isozymes in human cells.
Ebselen, 2-phenyl-1,2-benzoisoselenazol-3(2H)-one, is an isoselenazol well known for its antioxidant and anti-inflammatory properties (1, 24) and is widely used in laboratories as peroxide reducing antioxidant in in vivo models and has been proved in clinical trails against acute ischemic stroke (2, 25-31). We have previously shown that ebselen and its diselenide are substrates for mammalian TrxR and efficient oxidants of reduced Trx forming the ebselen selenol, the active form of ebselen with its hydrogen peroxide reductase activity (3a,3b). The mechanism of antioxidant action of ebselen, together with its diselenide, was mainly through its interactions with the mammalian TrxR and Trx, providing the electrons for the reduction of hydrogen peroxide from NADPH (3a, 3b, 32). In the present invention we have discovered that ebselen, however, is not a substrate of E. coli TxrR, but instead it is a competitive inhibitor for the reduction of thioredoxin with a Ki of 0.15 μM. E. coli mutants lacking a functional glutaredoxin system (glutathione reductase, GSH or glutaredoxin 1) were much more sensitive to inhibition by ebselen, which thereby will inhibit the essential enzyme ribonucleotide reductase (RNR) required for DNA synthesis. A main target of action of ebselen is the thioredoxin system. It follows that gram positive bacteria or other microorganisms lacking GSH will be particularly susceptible to ebselen. The present invention demonstrates that the well tolerated drug ebselen inhibits bacterial growth due to the large differences in structure and mechanism of the bacterial and mammalian thioredoxin reductases, establishing the drug as a novel chemotherapeutic principle.
It has been reported that ebselen inhibits bacteria growth with much higher sensitivity towards Staphylococcus aureus than E. coli (10, 33). However the mechanism behind this inhibition was not previously known. The present inventors have found that ebselen and its diselenide are strong inhibitors of E. coli TrxR. In bacterial inhibition experiments using mutant strains lacking the enzyme glutathione reductase (GR encoded by the gor gene) or glutathione (gshA− strain can not synthesize GSH) showed increased sensitivity towards ebselen. The interaction mechanism of ebselen and its diselenide with E. coli was studied showing the formation of a relative stable ebselen-TrxR complex at the active site of the enzyme. Interestingly, we found that the sulfur analogue of ebselen, ebsulfur (PZ25), and its disulfide were not inhibitors of the E. coli enzyme, but rather were substrates for the E. coli TrxR. However, as shown below, this is not the case for all bacterial enzymes since the Helicobacter pylori TrxR is inhibited.
Comparing the kinetic parameters of the interaction between the compounds and the two enzyme systems, provides better understanding of the chemical basis for the inhibition mechanism of ebselen and its diselenide towards the E. coli TrxR. This enhanced understanding of the principle chemical mechanism of ebselen diverse activity towards mammalian and E. coli TrxR is very important for the use of the drug and also for the development of effective antibiotic drugs based on same mechanism.
Furthermore, the finding that ebselen can inhibit E. coli TrxR leads us to a search for the new organoselenium compounds containing the basic structure of ebselen, to study their reactivity with E. coli thioredoxin reductase. We synthesized benzisoselenazol-3(2H)-ones and studied their reaction towards the thioredoxin reductase, to find out the relationship between the structure and reactivity. These compositions have, to varying extent, inhibitory effects on E. coli TrxR and bacterial growth, and therefore may be useful as antibiotics.
Different classes of benzisoselenazol-3(2H)-one compounds such as N-aryl (EbSe 7-10), N-unsubstituted (EbSe 6), N-alkyl (EbSe 2-4), N-2-pyridyl (EbSe 11 & 12) and N-4-pyridyl (EbSe 13) substituted benzisoselenazol-3(2H)-ones as well as bis-benzisoselenazol-3(2H)-ones (EbSe 14-16) were synthesized. Their inhibition effect on E. coli thioredoxin reductase (TrxR) was studied by thioredoxin dependent DTNB disulfide reduction assay in vitro. Detailed kinetic studies show that bisbenzisoselenazol-3(2H)-ones compounds (EbSe 14-16) inhibit TrxR at nanomolar concentrations while compounds EbSe 7-10, 12-13, 2-4 and parent ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (EbSe 6) inhibit at micromolar concentrations. Other compounds did not inhibit E. coli TrxR. Tryptophan fluorescence measurements were carried out to follow the reaction of these compounds with reduced thioredoxin. Like ebselen, these compounds also rapidly oxidized reduced thioredoxin.
Different classes of benzisoselenazol-3(2H)-one-aryl (EbSe 1-5), unsubstituted (EbSe 6), alkyl (EbSe 7-8), 2-pyridyl (EbSe 9 & 10) and 4-pyridyl (11) substituted benzisoselenazol-3(2H)-ones, bisbenzisoselenazol-3(2H)-ones (EbSe 12-14), 7-azabenzisoselenazol-3(2H)-one (EbSe 17), selenamide (EbSe 20) and bis(2-carbamoyl)phenyl diselenide (EbSe 21) have various levels of antibiotic activity, and for example inhibit bacterial (e.g., E. coli) thioredoxin reductase (TrxR). Detailed kinetic studies show that bisbenzisoselenazol-3(2H)-ones compounds (EbSe 12-14) inhibit TrxR at nanomolar concentrations while compounds EbSe 6, 2, 9, 11-13, 17, and parent ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (EbSe 1) inhibit at micromolar concentrations. Like ebselen, these compounds also rapidly oxidized reduced thioredoxin. See, U.S. Pat. No. 8,592,468, expressly incorporated herein by reference.
U.S. Pat. No. 8,592,468, expressly incorporated herein by reference discloses that benzisoselenazol-3(2H)-one and bisbenzisoselenazol-3(2H)-one derivatives were tested as potential. E. coli TrxR inhibitors, Measured IC50 and Ki values (Table 1) indicate that the compounds EbSe 1-4, 10-14 are potent inhibitors for E. coli TrxR. The presence of covalent bond between selenium and nitrogen is important for the biological property of ebselen derivatives. The inhibition effect of selenamide (EbSe 20) was tested, which also possess direct Se—N bond. However, it has reduced inhibition effect than ebselen derivatives. Other derivatives EbSe 5-9 did not show significant inhibition on E. coli TrxR.
The oxidation properties of benzisoselenazol-3(2H)-one derivatives on reduced E. coli Trx-(SH)2 were studied. Ebselen is reported as superfast thioredoxin oxidant, (32) and hence, used as the reference to compare the oxidant property of other compounds. The change of fluorescence intensity of 0.2 μM Trx-(SH)2 by mixing with 0.2 μM benzisoselenazol-3(2H)-one show that all of the ebselen derivatives can oxidize the reduced Trx as the reference compound ebselen under identical conditions.
From the data shown in Table 1, it can be clearly seen that the substitution at the nitrogen atom of the benzisoselenazol-3(2H)-one ring has a significant effect on the inhibition of TrxR. The substitution of benzisoselenazol-3(2H)-one linked by alkyl chains (EbSe 12-14) has stronger inhibitory effect than unsubstituted (EbSe 6), alkyl (EbSe 7-8), aryl (EbSe 1-5), 2-pyridyl (EbSe 9-10) substituted ones, and also than compound EbSe 11 where the condensed benzene ring of benzisoselenazol-3(2H)-one is replaced by a pyridine ring. Compounds EbSe 12-14 show similar inhibitory effect irrespective of substitution at the second nitrogen atom and the number of alkyl chains between the two nitrogen atoms. From this observation it seems the second heteroatom nitrogen present in these compounds seems to important characteristic for their strong inhibition. Comparison of EbSe 6-8 show there is no inhibition when hydrogen is substituted by methyl (EbSe 6) or tert-butyl (EbSe 7) group. On the other hand comparison of between EbSe 1, 10 and 11 indicates that modification of 2-phenyl-1,2-benzisoselenazol-3(2H)-one to 2-pyridyl benzisoselenazol-3(2H)-one or 7-azabenzisoselenazol-3(2H)-one does not have significant effect. Also inhibition is not much affected by the substitution of phenyl group attached to the nitrogen of benzisoselenazol-3(2H)-one. Selenamide EbSe 20 has by far less inhibition effect than the ebselen derivatives though direct Se—N bond present. It indicates the presence of five membered heterocyclic ring in addition to direct Se—N bond in the basic ebselen structure seems to so essential for their biological activities.
Bacterial TrxR is potent target for antibiotics development, in particular for the bacteria lacking glutathione system (see, US 2014/0088149; 2011/0288130; and 2009/0005422, expressly incorporated herein by reference). Here E. coli DHB4 strains wt, gshA−, gor−, oxyR− were used as the model to test the antibiotics activity of these ebselen derivates. The MICs of these compounds were list in Table 1. Corresponding to the inhibition capacity of E. coli TrxR, ebselen derivatives EbSe 1-4 and EbSe 11-14 had strong ability to inhibit the bacterial growth. E. coli wt strain, strains gshA− or gor− which lost a functional glutathione system show more sensitive to ebselen derivatives EbSe 1-4 and 11, suggesting glutathione system play a critical roles in the protection of bacteria from these compound. Whereas, all these strains exhibited the same sensitivity to EbSe 12, 14. This observation was verified by the further GPx activity measurement of these compounds (Table 1). The compounds EbSe 1-4 and 11 can react with glutathione and then induce the consumption of NADPH. In contrast, no GPx activity was observed for compound EbSe 12.
The inhibition of mammalian TrxR and the cytotoxicity of these ebselen derivates (Table 1) was also examined. Ebselen EbSe 12-14 was the strongest inhibitor for mammalian TrxR with a nanomolar inhibitory level, and also showed toxicity for mammalian cells. Ebselen EbSe 4 had some activity to inhibit mammalian TrxR, but it was one of the least reagent among these compounds. This result may be explained by the property that the compound is the best reagent to react with glutathione. The other ebselen derivatives EbSe 1-3 did not inhibit mammalian TrxR and were less toxic reagents for the mammalian cells.
In summary; different classes of benzisoselenazol-3(2H)-one substituted compounds were found to exhibit different antibiotic properties because of their inhibition capacity on bacterial thioredoxin reductase. Generally, the -aryl, 2-pyridyl and 4-pyridyl substituted compounds possess a good inhibition ability to bacterial TrxR as well as the strong inhibition on bacterial growth and less toxicity. But the more substitution such as with chloro, carbono, or nitro substitution can alter antibiotic property. The Se—N bond the structure is essential for the inhibition of bacterial TrxR as well as the inhibition of bacteria. Benzisoselenazol-3(2H)-one-unsubstituted or alkyl substituted compounds do not have the ability to inhibit bacterial TrxR. Bisbenzisoselenazol-3(2H)-ones have the strong inhibition for bacteria but also have the strongest toxicity for the mammalian cells.
Results
Ebselen and Ebselen Diselenide are Strong Competitive Inhibitors Towards E. coli TrxR.
When ebselen and ebselen diselenide are directly added in the solutions of E. coli TrxR and NADPH, no oxidations of NADPH were found. This is in line with the known fact that E. coli TrxR is strictly specific towards E. coli Trx. Further we examined the effect of ebselen in the reduction of disulphide by E. coli Trx and TrxR using both DTNB and insulin as substrates. Ebselen and its diselenide strongly inhibited the E. coli TrxR reduction towards E. coli Trx in a typical DTNB coupled assay. The same inhibition patterns are also shown for ebselen and ebselen diselenide in the insulin reduction assays (data not shown),
E. coli Trx largely increases the rate of reduction of ebselen and ebselen diselenide by mammalian TrxR (3, 32). Direct reduction of ebselen and the diselenide reduced E. coli Trx also were observed by fluorescence spectroscopy and the second-order rate constants were determined to be 2×107 M−1s−1 and 1.7×103 M−1s−1, respectively (32). Thus ebselen and the diselenide are targeting the E. coli TrxR rather than the E. coli Trx.
The degree of inhibition caused by ebselen appears to depend on the concentrations of Trx and ebselen. An increase in [Trx] at constant [EbSe] decreases the degree of inhibition and an increase in [EbSe] at constant [Trx] increases the degree of inhibition, showing a typical competitive inhibition towards the TrxR. A series of Lineweaver-Burk plots of the initial rate for the reduction of DTNB in the presence of ebselen and ebselen diselenide gave a typical pattern of competitive inhibitions. The dissociation constants Ki for the ebselen-TrxR and ebselen diselenide-TrxR complexes derived from the slopes [(KM/kcat)(1+[I]/Ki)] were 0.14±0.05 μM and 0.46±0.05 μM, respectively.
TABLE 2Kinetic parameters determined for ebselen, its diselenide and their sulphur analogues with mammalian and E. coli TrxR.Mammalian TrxRE. coli TrxRkcatKMkcat/KMkcatKMkcat/KMCompounds(min−1)(μM)(μM−1min−1)(min−1)(μM)(μM−1min−1)EbSea5882.5235Inhibitor with Ki = 0.15 ± 0.05 μM(EbSe)2b79402Inhibitor with Ki = 0.46 ± 0.03 μMEbS14002.55607002.5280(EbS)21500473210027.63.63afrom ref (3); bfrom ref (32).
Ebselen inhibits the growth of E. coli strains and more sensitive towards gor− and grxA− mutants.
Since ebselen was a potent inhibitor of E. coli thioredoxin reductase we examined whether strains lacking components of the GSH-glutaredoxin reducing pathway (FIG. 6) would be more sensitive to the drug. Thus we examined the sensitivity of gor− and gshA− mutants to ebselen, which reside heavily on the TrxR reducing pathway. Wild type bacteria were more resistant than gor− and gshA− strains with gor− and gshA− strains being the most sensitive. This indicates that elimination of parts of the GSH pathway renders cells sensitive to ebselen. The explanation could be that ebselen inhibits TrxR or the thioredoxins, or is eliminated in cells by GSH. The sensitivity of strain trxA−C− was similar, if not less, than that of the wild type, suggesting that the two E. coli thioredoxins were not primary targets for the compound. However ebselen may be affecting a thioredoxin 1 related function as the gshA−trxA− strain was more sensitive to the compound. In rich LB liquid cultures, resistance could additionally be associated with GSH from the culture medium which binds and neutralizes ebselen. The sensitivity to ebselen was increased in minimal media where gor− and gshA− strains hardly grew in its presence.
Sensitivity of Pathogenic Bacteria to Ebselen
Glutathione system is lacking and thus thioredoxin system is critical in many bacteria including some important pathogenic bacteria, such as methicillin resistant Staphylococcus aureus, Helicobacter pylori, Mycobacterium tuberculosis etc (36-40). Based on our principle that ebselen can target thioredoxin system in glutathione deficient bacteria, ebselen is the potential drug for inhibition of these bacterial. As also shown in reference 10, methicillin resistant Staphylococcus aureus, Bacillus subtilis are quite sensitive to ebselen. We also investigated Mycobacterium tuberculosis sensitivity on ebselen, the test was done in the radiometric BACTEC 460 system. As shown in Table 3, several multridrug resistant Mycobacterium tuberculosis strains are sensitive to ebselen. The medium contains 5 g/l of albumin or 70 μM which will bind ebselen. Ebselen at 10 mg/1l is 26 μM. The albumin free SH groups are about 50% or 35 μM. Therefore the MIC is dependent upon albumin saturation and probably lower than 20 mg/l.
We also investigated the inhibition of ebselen on H. pylori. For two macrolide sensitive strains, the minimal bactericidal concentration (MBC) are 3.125 and 6.25 μg/ml, for macrolide resistant strains, the MBC is 12.5 μg/ml. Taken together, our results strongly support that the inhibition of ebselen on these glutathione deficient bacteria is due to the oxidization of thioredoxin system by ebselen.
TABLE 3Sensitivity of MDR Mycobacterium tuberculosis to ebselenSensitivity to ebselen (μg/ml)StrainAb-res80402010H37RvSSSSRPanel3:24MDRSSSRBTB 98-310MDRSSSRS: sensitive to rifampicin as positive control (no growth); R: resistant.
TABLE 4Bactericidal effects of ebselen on Helicobacter pyloriSensitivity toSensitivity to ebselen (μg/ml)StrainMacrolide100502512.56.253.1251.560.78MS G6SSSSSSSRRMS G142SSSSSSRRRMR G162RSSSSRRRRMR G193RSSSSRRRRS: sensitive; R: resistant.E. coli TrxR Inhibition by Ebselen Derivates
All the benzisoselenazol-3(2H)-one and bisbenzisoselenazol-3(2H)-one derivatives were tested as potential E. coli TrxR inhibitors by standard DTNB assay. IC50 values were calculated by following the activity of TrxR reducing DTNB by NADPH at 412 nm. The reactions were started by adding 1 mM DTNB to the mixture of 100 nM TrxR, 2 μM Trx, 240 μM NADPH, and different concentration of inhibitor (1-40 μM). For determining the inhibition constants (Ki), indicated amount of inhibitor was mixed with total volume 500 μL containing 1 mM DTNB, 240 μM NADPH, fixed thioredoxin concentration (1 or 2 or 4 μM) and buffer containing 50 mM Tris-Cl, 2 mM EDTA, pH 7.5. The reactions were started by adding 6 nM TrxR at room temperature. Inhibition constants (Ki) for all the compounds were measured from Dixon plot, which plots 1/v versus [I]=A412/min, I=Inhibitor concentration). Measured IC50 and Ki values (Table 1) indicate that the compounds EbSe 6-9, 12-16 are potent inhibitors for E. coli TrxR. The presence of covalent bond between selenium and nitrogen is so important for the biological property of ebselen derivatives. Other derivatives did not show significant inhibition on E. coli TrxR.
Oxidation E. coli Trx-(SH)2 by Ebselen Derivates
Oxidant property of benzisoselenazol-3(2H)-one derivatives on reduced E. coli Trx-(SH)2 were studied by fluorescence spectroscopy. This property was chosen to follow the reaction of Trx with benzisoselenazol-3(2H)-one derivatives since E. coli Trx-(SH)2 has 3-fold higher tryptophan fluorescence than Trx-S2. Ebselen is reported as superfast thioredoxin oxidant[32] and hence, used as the reference to compare the oxidant property of other compounds. The change of fluorescence intensity of 0.2 μM Trx-(SH)2 by mixing with 0.2 μM benzisoselenazol-3(2H)-one show that they all can oxidize the reduced Trx as the reference compound ebselen under identical conditions.
Correlation Between the Structure and their Inhibition
From the data shown in Table 1, it can be clearly seen that the substitution at nitrogen atom of benzisoselenazol-3(2H)-one ring have significant effect on the inhibition of TrxR. The substitution of benzisoselenazol-3(2H)-one linked by alkyl chains (14-16) has stronger inhibitory effect than unsubstituted (EbSe 6), alkyl (EbSe 2-4), aryl (EbSe 7-10), 2-pyridyl (EbSe 11-12) and 4-pyridyl (EbSe 13) substituted ones. Compounds EbSe 14-16 show similar inhibitory effect irrespective of substitution at the second nitrogen atom and the number of alkyl chains between the two nitrogen atoms. From this observation it seems the second heteroatom nitrogen present in these compounds seems to important characteristic for their strong inhibition. Comparison of EbSe 2-4 show there is no inhibition when hydrogen is substituted by methyl (6) or tert-butyl (7) group. On the other hand comparison of EbSe 6, 12 and 13 indicates that modification of the 2-phenyl-1,2-benzisoselenazol-3(2H)-one into an N-2-pyridyl benzisoselenazol-3(2H)-one or an N-4-pyridyl benzisoselenazol-3(2H)-one does not have a significant effect. Also inhibition is not much affected by the substitution of phenyl group attached to the nitrogen of benzisoselenazol-3(2H)-one.
Inhibition of Bacterial Growth by Ebselen Derivates
Bacterial TrxR is potent target for antibiotics development, in particular for the bacteria lacking glutathione system. Here E. coli DHB4 strains wt, gshA−, gor−, oxyR− were used as the model to test the antibiotics activity of these ebselen derivatives corresponding to the inhibition capacity of E. coli TrxR, ebselen derivates EbSe 6-9 and 13-16 had strong ability to inhibit the bacterial growth. E. coli wt strain, strains gshA− or gor− which lost a functional glutathione system show more sensitive to ebselen derivates EbSe 6-9 and 13, suggesting glutathione system play a critical roles in the protection of bacteria from these compound. Whereas, all these strains exhibited the same sensitivity to EbSe 14, 16.
Inhibition of H. pylori TrxR and H. pylori Strains by PZ-25 (Ebsulfur).
H. pylori TrxR activity was inhibited by 4, 20, and 40 μM of PZ-25 by insulin reduction assay. Consistent with the inhibition of H. pylori TrxR activity, H. pylori strains were shown to be sensitive to ebsulfur. For NCTC11637 strain, the MIC for ebselen, PZ-25, metronidazole was 3.13, 1.56, and 0.78 μg/ml respectively. For strain YS-16, The MIC for ebselen, PZ-25, metronidazole was 3.13, 0.39, 6.25 μg/ml respectively.